1. Field of the Invention
The present invention relates to methods and devices for separating analytes in a solution using microcapillary devices.
2. Background
Various types of separation techniques are available for separating analytes in a solution. For example, flat-plate electrophoretic gel methods have been used to separate DNA components for the base sequencing of DNA. This method typically involves a gel that is sandwiched between two glass plates. DNA in the gel sample migrates electrophoretically by applying a voltage across both ends of the glass plates. Other methods for separating biological samples, such as DNA, include capillary electrophoretic devices. In these devices, the sample is placed in a gel. The gel is placed in capillaries typically made of silica or quartz. A sample migrates electrophoretically when a voltage is applied across the length of the capillary or capillary array assembly. Other methods and devices for separating analytes are discussed in U.S. Pat. No. 6,001,266, the disclosure of which is hereby incorporated by reference in its entirety.
In silica or quartz microcapillaries, ionized silanol groups (Si—OH) generally congregate on the inner surface of the capillary wall. The negatively charged surface can be balanced by a sheath of counter ions, such as H3O+ from the buffer, to form an electric double layer. When an electric field is applied along the length of the microcapillary, the positive ions in the diffuse part of the liquid migrate to the electrode of opposite polarity (generally the cathode). Liquid within the electric double layer is entrained by the migrating ions. The resulting “plug-like” flow, or electroosmotic flow, transports analytes dissolved in the liquid. Charged analytes can then separate electrophoretically. Depending on charge, solvation, and mass, the electric field will cause the analytes to migrate at different rates as they move toward the anode.
Biomolecules, such as DNA strands and proteins, generally carry a negative charge. At greater pH values, such as a pH value of more than three, the electrophoretic force on an anion towards the anode is generally weaker than the drag force of the buffer's electroosmotic flow toward the cathode. At lower pH values, such as lower than three, the anions generally migrate toward the anode.
Because the above separation process in a microcapillary depends on charge, solvation, and mass, certain analytes in a solution may not separate from one another. For example, an analyte that is twice as large and has twice as much charge as another analyte may travel at the same speed. That is, an increase in friction due to an increase in size of one analyte compared to another may slow its speed; however, if the analyte also has an increased charge, its speed may be the same as the smaller analyte with a lower charge.